The SPARC protein (secreted protein, acidic and cystein-rich protein) also known as osteonectin or BM40, is a secreted glycoprotein, highly distributed in human and non-human tissue, which functions and effects are wide and various. It has been found that it interacts with extracellular matrix components, with growth factors, with cytokines and with the expression of matrix metalloproteinases.
The SPARC protein has been initially described by Ledda M. F. et. al. (Ledda, M. F., Adris, S., Bravo, A. I., Kairiyama, C., Bover, L., Chernajovsky, Y., Mordoh, J., and Podhajcer, O. L., Suppression of SPARC expression by antisense RNA abrogates the tumorigenicity of human melanoma cells. Nat Med, 3: 171-176, 1997) as having a central role in the malignity of human melanoma. Subsequent studies showed that SPARC over-expression is associated to the malignant progression of various tumor types (Porte, H., Triboulet, J. P., Kotelevets, L., Carrat, F., Prevot, S., Nordlinger, B., DiGioia, Y., Wurtz, A., Comoglio, P., Gespach, C., and Chastre, E. Overexpression of stromelysin-3, BM-40/SPARC, and MET genes in human esophageal carcinoma: implications for prognosis. Clin Cancer Res, 4: 1375-1382, 1998). The SPARC protein is highly expressed both in endothelium and activated fibroblasts of in vivo tumors (Lane, T. F. and Sage, E. H. The biology of SPARC, a protein that modulates cell-matrix interactions. Faseb J, 8: 163-173, 1994).
Human SPARC promoters (Hafner, M., Zimmermann, K., Pottgiesser, J., Krieg, T., and Nischt, R. A purine-rich sequence in the human BM-40 gene promoter region is a prerequisite for maximum transcription. Matrix Biol, 14: 733-741, 1995), murine SPARC promoters (McVey, J. H., Nomura, S., Kelly, P., Mason, I. J., and Hogan, B. L. Characterization of the mouse SPARC/osteonectin gene. Intron/exon organization and an unusual promoter region. J Biol Chem, 263: 11111-11116, 1988) and bovine SPARC promoters (Young, M. F., Findlay, D. M., Dominguez, P., Burbelo, P. D., McQuillan, C., Kopp, J. B., Robey, P. G., and Termine, J. D. Osteonectin promoter. DNA sequence analysis and S1 endonuclease site potentially associated with transcriptional control in bone cells. J Biol Chem, 264: 450-456, 1989) have been cloned and characterized. The comparison between these promoters shows that, similarly to what it is observed at gene level, a high sequence homology occurs.
The structure of the human SPARC promoter is shown in FIG. 1, wherein the first exon, the GGA1 and GGA2 boxes, the 10 nucleotide Inter-CGA region separating them and the TATA non-consensus sequence are depicted. The human SPARC promoter lacks a TATA consensus box (Breathnach, R. et al., Organization and expression of eucaryotic split genes coding for proteins, Annu Rev Biochem, 50: 349-383, 1981) but contains a so-called TATA-like element that shares some bases with the conventional sequence. The promoter has two GCA1 and GCA2 boxes, of which the GCA1 box exhibits a great similarity between the human and bovine species.
Hafner et al. observed that the GCA1 box is necessary and sufficient for obtaining a maximum transcriptional activity, while the spacing element separating the two CGA boxes has a negative effect on its expression (Hafner M. et al., 1995). It is important to note that this group has demonstrated that, in humans, the promoter region containing only the CGA boxes is not sufficient by itself to confer expression specificity in different cell lines. Dominguez et. al. described the region between bases −504 to +11 of the bovine promoter as a positive element for SPARC transcription in fetal bovine cells. This fragment also confers specific expression, showing higher activity in cells with a higher expression level of SPARC mRNA (Dominguez, P., Ibaraki, K., Robey, P. G., Hefferan, T. E., Termine, J. D., and Young, M. F. Expression of the osteonectin gene potentially controlled by multiple cis-and trans-acting factors in cultured bone cells. J Bone Miner Res, 6: 1127-1136, 1991). They also observed that only the GC box (which is a common element in many promoter regions and their consensus sequence is GGGCGG, may be present in more than one copy. It is located between the −40 to −100 bp) and the GCA1 box are not sufficient for the maximum SPARC expression in bovine bone cells, and that the region located between the bases −927 to −504 produces a dramatic inhibition of transcription.
The gene therapy potentially represents one of the most important developments taking place in medicine. In order to modify a specific cell or tissue type, therapeutic genes have to be efficiently administered to the cell so that the gene expresses in the appropriate level and for a sufficient amount of time. Two types of strategy are being applied for DNA supply to cells, these are by viral and non-viral vectors. Even though a great number of virus destined to gene transfer have been developed, the major interest has been centered in retrovirus, adenovirus, adeno-associated virus and herpes simplex virus type 1. The first generation adenovirus are defective in E1A protein, hence they do not replicate. The early E1A protein is the first protein that produces the viral DNA inside the cell. E1A has many functions such as helping other viral proteins to be produced and stimulating the cell growth by bonding Rb and releasing E2F, facilitating viral transcription and replication. Even though those E1A protein defective adenovirus were successfully used as vectors in cancer pre-clinical models, the same results were not achieved when used in clinical trials, being its low in vivo transduction capacity one of the major problems (Vile, R. Cancer gene therapy—new approaches to tumour cell killing. J Gene Med, 2: 141-143, 2000).
One way to overcome this drawback has been the creation of a new generation of vectors capable of conditionally replicating in the tumor environment; these vectors are called CRAd (Conditionally Replicative Adenovirus or Oncolytic adenovirus). CRAds are constructed by modifying the adenoviral genome in order to regulate the expression of E1A protein with a promoter that is specifically active in the required tissue or cell type, in such a way to prevent damage to surrounding tissues.
In the last years, several research groups have devoted to the recombinant adenovirus construction. This way, some of the viral genes that had been removed in the past are being re-inserted again given that they enhance viral replication. That is the case of the E3 region. E3 is a viral DNA fragment encoding 9 proteins, the main function of which is the inhibition of cell death induced by the host immune response. Among the 9 proteins, ADP (Adenoviral Death Protein) stands out, has a contradictory function when compared to their E3 mates, because it promotes the late cell lysis in the viral infection cycle to allow for the release of mature virions to the cell microenvironment. Cells infected with an non-expressing ADP adenovirus have been shown to remain viable for a longer time than cells infected with the wild type adenovirus (Tollefson, A. E. et al. The E3-11.6-kDa adenovirus death protein (ADP) is required for efficient cell death: characterization of cells infected with adp mutants, Virology, 220: 152-162, 1996; Tollefson, A. E et al., The adenovirus death protein (E3-11.6K) is required at very late stages of infection for efficient cell lysis and release of adenovirus from infected cells, J Virol, 70. 2296-2306, 1996; Kruyt, F. A. et al., A new generation of conditionally replicating adenoviruses: pairing tumor selectivity with maximal oncolysis, Hum Gene Ther, 13: 485-495, 2002. ).
One of the most attractive ways of gene therapy is the use of suicide genes. The basis of the system consists of introducing a gene encoding an enzyme with the capacity of metabolizing a non-toxic prodrug turning it into a toxic drug. One of the mostly used genes is the Herpes simplex virus thymidine kinase or HSV/TK, which codifies for an enzyme capable of phosphorilating the prodrug acyclovir/ganciclovir (commonly used antiviral for viral infections), a guanosine analogue. In its phosphorilated form, the anti-herpetic agent is incorporated to the DNA molecule, avoiding its duplication and causing cell death (Moolten, F. L., Drug sensitivity (“suicide”) genes for selective cancer chemotherapy, Cancer Gene Ther, 1: 279-287, 1994). The neighbor non-transduced tumor cells may also be eliminated by the so-called bystander effect, which allows the toxic metabolites to be transferred from an affected cell to a non-affected cell.
A tumor is formed by tumor cells, fibroblasts and endothelial cells. This is why an effective therapy with viral vectors requires the virus to be able to replicate in these three cell types, which are responsible of tumor progression. Given that SPARC is over-expressed in all these cell types, it represents a good candidate for the construction of a CRAd, such that the SPARC promoter drives the gene of interest, such as an E1A gene and eventually another therapeutic gene. In this sense, the tumor cells would be eliminated by the replication of the virus itself or by the action of the toxic drug produced in the tumor environment.